【作者】 王静；

【导师】 张志红；

【作者基本信息】 山西医科大学 ， 公共卫生（专业学位）， 2019， 硕士

【摘要】 目的:研究交通相关PM_2.5不同组分对哮喘大鼠肺部炎症的影响以及STAT5、Foxp3基因甲基化调控JAK2/STAT5/Foxp3信号通路在哮喘加重中的作用,以探讨PM_2.5不同组分诱导哮喘加重的机制。方法:90只雄性SD大鼠随机分为9组:生理盐水对照组,OVA组,OVA+PM_2.5组（3mg/kg.bw）,OVA+PM_2.5水溶成分低剂量组（1.8mg/kg.bw）,OVA+PM_2.5水溶成分高剂量组（7.2mg/kg.bw）,OVA+DMSO溶剂对照组,OVA+PM_2.5有机成分低剂量组（0.6mg/kg.bw）,OVA+PM_2.5有机成分高剂量组（2.4mg/kg.bw）,OVA+PM_2.5有机成分高剂量（2.4mg/kg.bw）+5-Aza组。通过卵清蛋白OVA腹腔注射致敏、雾化吸入激发构建大鼠哮喘模型,激发后予以不同剂量PM_2.5和PM_2.5水溶及有机组分气管滴注染毒,每三天一次,共五次,5-Aza在每次染毒前15 min予以腹腔注射。末次染毒后处死大鼠,收集支气管肺泡灌洗液（BALF）,肺组织病理切片HE染色观察病理改变。焦磷酸测序检测肺组织STAT5、Foxp3基因启动子区CPG岛各位点甲基化水平。Western Blot法检测肺组织JAK2、STAT5、P-STAT5、Foxp3蛋白相对表达量。免疫组织化学法检测肺组织P-STAT5、Foxp3蛋白表达水平。流式细胞术检测外周血Treg细胞比例。ELISA法检测BALF中IL-10和TGF-β1含量。结果:（1）PM_2.5及其水溶、有机成分暴露可使细支气管周围有明显的炎性细胞的浸润,管腔内杯状细胞增生,粘液分泌增多。5-Aza处理后细支气管周围的炎症细胞明显减少。（2）与生理盐水对照组和OVA组相比,PM_2.5及PM_2.5水溶成分低、高剂量暴露组肺组织STAT5基因位点均值甲基化水平明显降低（P<0.05）。与OVA+DMSO溶剂对照组相比,PM_2.5有机成分低、高剂量暴露使肺组织Foxp3基因位点均值甲基化水平明显升高（P<0.05）。5-Aza处理后Foxp3基因位点均值甲基化水平明显减低（P<0.05）。（3）与生理盐水对照组和OVA组相比,PM_2.5暴露使肺组织JAK2蛋白表达水平明显升高（P<0.05）,STAT5、P-STAT5蛋白表达水平明显降低（P<0.05）;PM_2.5水溶成分低、高剂量暴露使肺组织STAT5、Foxp3蛋白表达水平明显比OVA组降低（P<0.05）。与OVA+DMSO溶剂对照组相比,PM_2.5有机成分低剂量暴露使肺组织JAK2蛋白表达水平明显升高（P<0.05）,STAT5、P-STAT5、Foxp3蛋白表达水平明显降低（P<0.05）。5-Aza处理后STAT5、P-STAT5蛋白表达水平明显升高（P<0.05）。（4）与生理盐水对照组和OVA组相比,PM_2.5水溶成分高剂量暴露后外周血中Treg细胞比例明显降低（P<0.05）;与OVA+DMSO溶剂对照组相比,PM_2.5有机成分低剂量暴露后外周血中Treg细胞比例明显下降（P<0.05）。（5）与生理盐水对照组、OVA组、OVA+PM_2.5组相比,PM_2.5水溶成分高剂量暴露后BALF中IL-10的含量明显升高（P<0.05）。PM_2.5暴露使BALF中TGF-β1表达明显比OVA组和OVA+PM_2.5水溶成分低剂量组高（P<0.05）;与OVA+DMSO溶剂对照组相比,PM_2.5有机成分低、高剂量暴露使BALF中TGF-β1表达明显升高（P<0.05）。结论:（1）PM_2.5及其水溶和有机组分均可加重大鼠哮喘发作,肺部炎症加重。（2）STAT5和Foxp3基因甲基化参与调控PM_2.5及其水溶和有机组分引起的哮喘大鼠肺脏STAT5-Foxp3蛋白表达。（3）JAK2-STAT5-Foxp3信号通路参与调控PM_2.5及其有机组分诱发哮喘加重的机制,STAT5-Foxp3信号通路参与调控PM_2.5水溶组分诱发哮喘加重的机制。（4）PM_2.5及其水溶组分和有机组分均可减少外周血中Treg细胞比例,促进肺泡灌洗液中IL-10、TGF-β1细胞因子的释放。更多还原

【Abstract】 Objective:This study is to observe the effects of different components of traffic-related PM_2.5.5 on the lung inflammation and the role of STAT5/Foxp3 gene methylation in regulation of JAK2/STAT5/Foxp3 in asthma exacerbation,and to explore the mechanism of asthma exacerbation induced by different components of PM_2.5.Methods:Ninety male SD rats were randomly divided into nine groups:saline control group,OVA group,OVA+PM_2.5.5 group（3 mg/kg）,OVA+low dose of PM_2.5.5 water-soluble extracts group（1.8mg/kg）,OVA+high dose of PM_2.5.5 water-soluble extracts group（7.2mg/kg）,OVA+DMSO group,OVA+low dose of PM_2.5.5 organic extracts group（0.6mg/kg）,OVA+high dose of PM_2.5.5 organic extracts group（2.4mg/kg）,OVA+high dose of PM_2.5.5 organic extracts（2.4mg/kg）+5-Aza group.Rats were sensitized by intraperitoneal injections of ovalbumin and challenged by inhaling ovalbumin.After challenge,rats were administrated intratracheally with different doses of PM_2.5.5 and PM_2.5water soluble and organic components once every three days,five times in total,5-Aza was injected intraperitoneally 15 mins before each exposure.The rats were sacrificed after the last exposure,and bronchoalveolar lavage fluid（BALF）was collected.The pathological changes were observed by HE staining of lung tissue pathological sections.The methylation levels of CpG promoter loci of STAT5 and Foxp3 genes in lung tissue were detected by pyrosequencing method.The protein expressions of JAK2,STAT5,P-STAT5and Foxp3 in lung tissue were detected by Western Blot.The protein expression levels of P-STAT5 and Foxp3 in lung tissue were detected by immunohistochemistry.The ratio of Treg cells in peripheral blood were detected by flow cytometry.The levels of IL-10 and TGF-β1 in BALF were determined by ELISA.Results:（1）PM_2.5.5 and its water-soluble and organic components can cause obvious infiltration of inflammatory cells around the bronchioles and the increase of goblet cells in bronchocoel and mucus secretion.The inflammatory cells around the bronchioles were significantly reduced after 5-Aza treatment.（2）Compared with the saline control group and OVA group,the exposure of PM_2.5and the low and high doses of PM_2.5.5 water-soluble components significantly decreased the methylation levels of CpG promoter loci of STAT5 in lung tissue（P<0.05）.Compared with the OVA+DMSO group,the low and high doses of PM_2.5.5 organic components increased the methylation level of CpG promoter loci of Foxp3 in lung tissue（P<0.05）.The methylation level of Foxp3 gene was significantly lower after 5-Aza treatment（P<0.05）.（3）Compared with the saline control group and the OVA group,PM_2.5.5 exposure significantly increased the expression of JAK2 protein in lung tissue（P<0.05）,and the expression levels of STAT5 and P-STAT5 protein were significantly decreased（P<0.05）.The low and high dose of PM_2.5.5 water-soluble components exposure significantly decreased the expression of STAT5 and Foxp3 protein in lung tissue compared with OVA group（P<0.05）.Compared with the OVA+DMSO group,the low dose of organic components exposure increased the expression of JAK2 protein in lung tissue（P<0.05）,and the expression levels of STAT5,P-STAT5 and Foxp3 protein were significant decreased（P<0.05）.The expression levels of STAT5 and P-STAT5 protein in 5-Aza treatment group were significantly increased（P<0.05）.（4）Compared with the saline control group and OVA,the ratio of Treg cells in peripheral blood was significantly decreased after exposure to high-dose PM_2.5water-soluble components（P<0.05）.Compared with OVA+DMSO group,the low dose of organic components exposure decreased the ratio of Treg cells in peripheral blood（P<0.05）.（5）Compared with saline control group,OVA group and OVA+PM_2.5.5 group,the content of IL-10 in BALF was significantly increased after high dose of PM_2.5water-soluble components exposure（P<0.05）.PM_2.5.5 exposure significantly increased the expression of TGF-β1 in BALF by comparison with OVA group and OVA+PM_2.5water-soluble component group（P<0.05）.Compared with the OVA+DMSO group,the low and high doses of PM_2.5.5 organic components increased the expression of TGF-β1 in BALF（P<0.05）.Conclusions:（1）PM_2.5.5 and its water-soluble and organic extracts can aggravate asthma symptoms and lung inflammation of rats.（2）STAT5 and Foxp3 gene methylation are involved in the regulation of STAT5-Foxp3 protein expression in lungs of asthmatic rats induced by PM_2.5.5 and its water-soluble and organic components.（3）The JAK2-STAT5-Foxp3 signaling pathway is involved in the regulation mechanism of asthma exacerbation induced by PM_2.5.5 and its organic components.The STAT5-Foxp3 signaling pathway is involved in the regulation mechanism of asthma exacerbation induced by PM_2.5.5 and its water-soluble components.（4）PM_2.5.5 and its water-soluble components and organic components can suppress the proliferation and differentiation of Treg in peripheral blood and promote the release of IL-10 and TGF-β1 cytokines in bronchial alveolar lavage fluid.更多还原